Interest in the enzyme dihydrofolate reductase (DHFR) derives in part from its clinical relevance as a target enzyme of anti-folate drug therapy. The reductase is inhibited by methotrexate, used in the treatment of various types of cancer, and the bacterial enzyme is strongly and specifically inhibited by trimethoprim, which is used in combination with sulfa drugs for treating a variety of infectious diseases. Studies carried out during the past year have been aimed at elucidating the catalytic mechanism of this enzyme by carrying out NMR analyses on the binary complex formed by the enzyme and isotopically labeled dihydrofolate. These studies have been motivated by proposals that protonation of the bound dihydrofolate facilitates the subsequent hydride transfer reaction from NADPH to yield the reduced tetrahydrofolate product plus NADP+. Based on speculation that this protonation is occurring at the N-5 position of the pterion ring system, derivatives containing nitrogen-15 at the N-5 position and carbon-13 at the C-6 position have been synthesized. As a consequence of the limited solubility of dihydrofolate, labeled analogs have also been prepared, including the pentaglutamyl derivative of dihydrofolate, and dihydrobiopterin. A series of pH dependent studies carried out on the complexes formed between the labeled dihydrofolate and analogs and the beef liver dihydrofolate reductase suggest that the general model based on N-5 protonation may be incorrect. Further studies of this important question are in progress.